Two previously described transgenic mouse lines were used for FACS sorting of SOX+ and DCX+ cells: one line expressed GFP under the Sox2 promoter and the other line expressed DsRed under the Dcx promoter6,23. For each experiment, 10 hippocampi and SVZs (from coronal sections) were dissected and pooled from 6- to 8-week-old mice. Dissected tissue was chopped into ~1 mm3 pieces using sterile razor blades and dissociated by incubation for 30 minutes in a solution containing 0.01% papain (25 u/mg, Worthington Biochemicals), 0.1% neutral protease (0.5 u/mg, Roche), and 0.01% DNaseI (2788 u/mg, Worthington Biochemicals). The cell suspension was mixed with an equal volume of DMEM:F12 media (containing 1 mM L-glutamine and 10% fetal bovine serum) and filtered through a 70-µm nylon mesh, mixed with an equal amount of Percoll solution and pelleted (20,000 g for 30 minutes). Cellular debris was removed, and cells were spun down and dissolved in 2 ml DMEM:F12 plus N2. For FACS, GFP was excited with a 488-nm water-cooled argon laser; DsRed was excited with a 561-nm solid-state laser. The filters used were 530/30 nm for GFP and 630/22 nm for DsRed using a BD FACSvantage Diva system (BD Biosciences).
Extracted molecule
total RNA
Extraction protocol
RNA from 1,000 cells from each cell population was isolated with TRIzol reagent (Invitrogen), amplified and reverse transcribed using the SuperAmp protocol (Miltenyi).
Label
Cy3
Label protocol
Cy3 labelling according to Miltenzi Biotec undisclosed protocol.
Hybridization protocol
Cy3 labeled cDNAs were hybridized overnight for 17 hours at 65 degrees Celsius to Agilent whole mouse genome Oligo Microarrays 4 x 44K using Agilent's recommended hybridization chamber and oven
Scan protocol
Scanning done by Agilent Microarray scanning system
Data processing
Raw Cy3 data were normalized to the median and all values that were smaller than 1 were set to zero. All genes that had zero values in at least 11 of 12 arrays were deleted from the list, leaving 29,148 genes for analysis. Data were analyzed using the CARMA software suite (www.carmaweb.genome.tugraz.at/carma). The values were log2 transformed and quantile normalized. Expression differences were calculated over 3 replicates between 2 groups of experiments with the LIMMA package employing a modified t-statistic. Benjamini and Hochberg False discovery rate was used to calculate p-values. Final gene lists contain all genes with a p-value <=0.05. Expression values of genes that were represented twice or more on the array were averaged.
